Robert L. Flower

Soluble Mediators in Platelet Concentrates Modulate Dendritic Cell Inflammatory Responses in an Experimental Model of Transfusion.

The transfusion of platelet concentrates (PCs) is widely used to treat thrombocytopenia and severe trauma. Ex vivo storage of PCs is associated with a storage lesion characterized by partial platelet activation and the release of soluble mediators, such as soluble CD40 ligand (sCD40L), RANTES, and interleukin (IL)-8. An in vitro whole blood culture transfusion model was employed to assess whether mediators present in PC supernatants (PC-SNs) modulated dendritic cell (DC)-specific inflammatory responses (intracellular staining) and the overall inflammatory response (cytometric bead array). Lipopolysaccharide (LPS) was included in parallel cultures to model the impact of PC-SNs on cell responses following toll-like receptor-mediated pathogen recognition. The impact of both the PC dose (10%, 25%) and ex vivo storage period was investigated [day 2 (D2), day 5 (D5), day 7 (D7)]. PC-SNs alone had minimal impact on DC-specific inflammatory responses and the overall inflammatory response. However, in the presence of LPS, exposure to PC-SNs resulted in a significant dose-associated suppression of the production of DC IL-12, IL-6, IL-1α, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP)-1β and storage-associated suppression of the production of DC IL-10, TNF-α, and IL-8. For the overall inflammatory response, IL-6, TNF-α, MIP-1α, MIP-1β, and inflammatory protein (IP)-10 were significantly suppressed and IL-8, IL-10, and IL-1β significantly increased following exposure to PC-SNs in the presence of LPS. These data suggest that soluble mediators present in PCs significantly suppress DC function and modulate the overall inflammatory response, particularly in the presence of an infectious stimulus. Given the central role of DCs in the initiation and regulation of the immune response, these results suggest that modulation of the DC inflammatory profile is a probable mechanism contributing to transfusion-related complications.

Seroprevalence of Antibodies to Ross River and Barmah Forest Viruses: Possible Implications for Blood Transfusion Safety After Extreme Weather Events

AbstractnClimate change is predicted to increase the transmission of many vector-borne pathogens, representing an increasing threat to a safe blood supply. In early 2011, Australia experienced catastrophic rainfall and flooding, coupled with increased arbovirus transmission. We used Ross River (RRV) and Barmah Forest (BFV) viruses as test cases to investigate the potential risk posed to Australia’s blood supply after this period of increased rainfalln. We estimated the risk of collecting an infected donation as one in 2,500–58,000 for RRV and one in 2,000–28,000 for BFV. Climate change may incrementally increase the arbovirus threat to blood safety.

Procoagulant role of microparticles in routine storage of packed red blood cells: potential risk for prothrombotic post-transfusion complications

During routine storage, packed red blood cells (PRBC) undergo biochemical and morphological changes including loss of red blood cell (RBC) membrane asymmetry and release of microparticles (MPs) bearing phosphatidylserine (PS), a procoagulant phospholipid. This study investigated the association between PRBC storage duration, MP profile and procoagulant activity. Leukodepleted PRBC-supernatant (PRBC-SN; n=13) was prepared at weekly intervals throughout storage. Phospholipid-dependent procoagulant activity, assessed using a factor X-activated clotting time (XACT) assay, decreased throughout storage (p<0.0001), corresponding with increased procoagulant phospholipid content. As determined by flow cytometry, total numbers of MPs and of PS-bearing MPs increased by Day 28 of storage (p<0.01 and p<0.05, respectively, versus D1), and these MPs were predominantly RBC-derived (CD235+). Depletion of MPs from stored (Day 42) PRBC-SN using 0.22xa0μm filters reduced the number of PS-bearing MPs (p<0.01) but did not increase XACT clotting times. Furthermore, the reduction in procoagulant activity when lactadherin was used to block PS was not altered pre- or post-filtration of PRBC-SN. In conclusion, routine PRBC storage was associated with accumulation of MPs (particularly RBC-derived PS-bearing MPs) and ofxa0procoagulant phospholipids; however, depletion of PS-bearing MPs by 0.22xa0μm filtration did not reduce phospholipid-dependent procoagulant activity.

Numerical Investigation of Motion and Deformation of a Single Red Blood Cell in a Stenosed Capillary

It is generally assumed that influence of the red blood cells (RBCs) is predominant in blood rheology. The healthy RBCs are highly deformable and can thus easily squeeze through the smallest capillaries having internal diameter less than their characteristic size. On the other hand, RBCs infected by malaria or other diseases are stiffer and so less deformable. Thus it is harder for them to flow through the smallest capillaries. Therefore, it is very important to critically and realistically investigate the mechanical behavior of both healthy and infected RBCs which is a current gap in knowledge. The motion and the steady state deformed shape of the RBCs depend on many factors, such as the geometrical parameters of the capillary through which blood flows, the membrane bending stiffness and the mean velocity of the blood flow. In this study, motion and deformation of a single two-dimensional RBC in a stenosed capillary is explored by using smoothed particle hydrodynamics (SPH) method. An elastic spring network is used to model the RBC membrane, while the RBCs inside fluid and outside fluid are treated as SPH particles. The effect of RBCs membrane stiffness (kb), inlet pressure (P) and geometrical parameters of the capillary on the motion and deformation of the RBC is studied. The deformation index, RBCs mean velocity and the cell membrane energy are analyzed when the cell passes through the stenosed capillary. The simulation results demonstrate that the kb, P and the geometrical parameters of the capillary have a significant impact on the RBCs motion and deformation in the stenosed section.

Effect of age, gender and mannose-binding lectin (MBL) status on the inflammatory profile in peripheral blood plasma of Australian blood donors.

Transfusion of blood components has been associated with poor patient outcomes and, an overall increase in morbidity and mortality. Differences in the blood components arising from donor health, age and immune status may impact on outcomes of transfusion and transfusion-related immune modulation in recipients. The aim of this study was to investigate differences in inflammatory profile in donors and association with parameters including age, gender and deficiency status of pattern recognition molecule mannose-binding lectin (MBL). MBL level was determined by ELISA. Serum levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, and IFN-γ were examined by cytometric bead array (CBA). C-reactive protein (CRP) and rheumatoid factor (RF) were examined by immunoturbidimetry. This study demonstrated age was a parameter associated with the immune profile of blood donors, with significant increases in MCP-1 (p<0.05) and RF (p<0.05) and decreases in IL-1α evident in the older donors (61-76years). Significant gender-associated differences in MCP-1, IL-12 and CRP plasma levels in the blood donor cohort were also reported. There was no significant difference in the level of any inflammatory markers studied according to MBL status. This study demonstrated that age and gender are associated with inflammatory profile in donors. These differences may be a factor impacting on outcomes of transfusion.

Antibodies to Leptospira among blood donors in higher-risk areas of Australia: possible implications for transfusion safety

BACKGROUNDnLeptospirosis is one of the most common bacterial zoonoses worldwide, and clinical manifestations range from asymptomatic infection to acute febrile illness, multi-organ failure and death. Asymptomatic, acute bacteraemia in a blood donor provides a potential for transfusion-transmission, although only a single such case from India has been recorded. Human leptospirosis is uncommon in developed countries; however, the state of Queensland in Australia has one of the highest rates among developed countries, especially after increased rainfall. This study examined the prevalence of antibodies to Leptospira spp. in blood donors residing in higher-risk areas of Australia, to evaluate the appropriateness of current blood safety guidelines.nnnMATERIALS AND METHODSnPlasma samples collected from blood donors residing in higher-risk areas of Australia during 2009 and 2011 were included in the study. All samples were tested for the presence of antibodies to 22 leptospiral serovars using the microscopic agglutination test.nnnRESULTnNo sample had antibody titres suggestive of a current or recent infection, however, seven samples (1.44%, 95% CI: 0.38-2.50%) had titres suggestive of a past infection.nnnDISCUSSIONnThis study provides data that may support the appropriateness of current relevant donor selection policies in Australia. Given that the risk profile for leptospirosis is expanding and that the infection is likely to become more prevalent with climate change, this disease may become more of a concern for transfusion safety in the future.

Characterization of ENU-induced Mutations in Red Blood Cell Structural Proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants: THE In(Lu) PHENOTYPE AND KLF1 VARIANTS

KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population.

Investigation of red blood cell mechanical properties using AFM indentation and coarse-grained particle method

BackgroundRed blood cells (RBCs) deform significantly and repeatedly when passing through narrow capillaries and delivering dioxygen throughout the body. Deformability of RBCs is a key characteristic, largely governed by the mechanical properties of the cell membrane. This study investigated RBC mechanical properties using atomic force microscopy (AFM) with the aim to develop a coarse-grained particle method model to study for the first time RBC indentation in both 2D and 3D. This new model has the potential to be applied to further investigate the local deformability of RBCs, with accurate control over adhesion, probe geometry and position of applied force.ResultsThe model considers the linear stretch capacity of the cytoskeleton, bending resistance and areal incompressibility of the bilayer, and volumetric incompressibility of the internal fluid. The model’s performance was validated against force–deformation experiments performed on RBCs under spherical AFM indentation. The model was then used to investigate the mechanisms which absorbed energy through the indentation stroke, and the impact of varying stiffness coefficients on the measured deformability.xa0This study found the membrane’s bending stiffness was most influential in controlling RBC physical behaviour for indentations of up to 200 nm.ConclusionsAs the bilayer provides bending resistance, this infers that structural changes within the bilayer are responsible for the deformability changes experienced by deteriorating RBCs. The numerical model presented here established a foundation for future investigations into changes within the membrane that cause differences in stiffness between healthy and deteriorating RBCs, which have already been measured experimentally with AFM.

Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125u2009mmol/L sucrose: 500u2009mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.